Ilaria De Santis1, Michele Zanoni2, Sara Pignatta2, Pasquale Longobardi3, Anna Tesei4, Alessandro Bevilacqua5,6. 1. Interdepartmental Centre Alma Mater Research Institute on Global Challenges and Climate Change (Alma Climate), University of Bologna, I-40126, Bologna, Italy. 2. Biosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", I-47014, Meldola, Italy. 3. Hyperbaric Centre, I-48124, Ravenna, Italy. 4. Biosciences Laboratory, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", I-47014, Meldola, Italy. anna.tesei@irst.emr.it. 5. Advanced Research Center on Electronic Systems (ARCES) for Information and Communication Technologies "E. De Castro", University of Bologna, I-40125, Bologna, Italy. alessandro.bevilacqua@unibo.it. 6. Department of Computer Science and Engineering (DISI), University of Bologna, I-40136, Bologna, Italy. alessandro.bevilacqua@unibo.it.
Abstract
PURPOSE: RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells. METHODS: In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension. RESULTS: We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction. CONCLUSION: Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response.
PURPOSE: RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells. METHODS: In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension. RESULTS: We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction. CONCLUSION: Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response.
Authors: Rachel E Rigby; Lauren M Webb; Karen J Mackenzie; Yue Li; Andrea Leitch; Martin A M Reijns; Rachel J Lundie; Ailsa Revuelta; Donald J Davidson; Sandra Diebold; Yorgo Modis; Andrew S MacDonald; Andrew P Jackson Journal: EMBO J Date: 2014-02-10 Impact factor: 11.598